1. Field of the Invention
The present invention relates generally to methods for detecting target analytes in samples which may contain cross-reacting substances. More particularly, the present invention relates to immunoassays which minimize binding between a detecting antibody and the cross-reacting substance.
A wide variety of assay techniques exist for detecting the presence of a target analyte in a biological specimen. Of particular interest to the present invention, specific binding assays rely on detecting the analyte using a binding substance which reacts with the analyte in a highly selective manner. The use and specificity of such specific binding assays has been greatly enhanced by the availability of monoclonal antibodies. Monoclonal antibodies allow the performance of immunoassays which can detect particular epitope(s) on an analyte of interest in a highly specific manner.
The detection of certain analytes which are antigenically similar to other substances which may be present in a particular specimen, however, remains problematic. In some cases, it will be desirable to detect an analyte which differs only in a small region of the molecule when compared to other substances which may be present in the sample. For example, it will sometimes be necessary to distinguish between proteins which differ by as little as one amino acid in their sequences. Similarly, it may be necessary to distinguish between glycoproteins and carbohydrates which differ by as little as one sugar residue or in other very minor ways. In such cases, the availability of monoclonal antibodies which can distinguish between these very small differences is not assured.
One such instance involves the detection of creatine kinase isoenzymes which are released from tissue damaged as a result of acute myocardial infarction. It has been found that patients suffering chest pain and other symptoms of myocardial infarction who have inconclusive electrocardiograms may be diagnosed based on the presence of certain creatine kinase (CK) isoenzymes in their blood. While such CK isoenzymes are normally released in the blood as a result of muscle cell turnover, the amount of CK isoenzyme MB (CKMB) is substantially higher in the myocardium than in other muscle tissues. Thus, the release of CKMB in the blood is diagnostic of myocardial infarction.
Although conclusive in most instances, present CKMB assays require a post-symptom period of about six hours in order for diagnostically significant levels of CKMB to build up in the blood. Such a delay in diagnosis is usually unacceptable, particularly when the measurement is being performed in order to initiate and monitor certain thrombolytic therapies to open stenosed arteries.
CKMB measurement can provide a much more rapid diagnosis by specifically detecting certain isoforms of the enzyme. The CKMB isoenzyme exists in tissue as a dimer of the M subunit and B subunit, with each subunit having a carboxy terminal lysine. After release into blood, the lysines of both subunits of the tissue isoform are cleaved, with the tissue isoform having a half life of several hours.
It is thus possible to diagnose acute myocardial infarction based on the relative amounts of the tissue and blood isoforms of CKMB. That is, individuals having elevated levels of the tissue isoform relative to the blood isoform can be assumed to have recently suffered significant tissue damage. Since CKMB is found at high levels in the myocardium, such levels will confirm a diagnosis of myocardial infarction, particularly when combined with the other symptoms which occasioned the test in the first place.
For these reasons, it would be desirable to provide improved specific binding assays, particularly immunoassays, which are capable of distinguishing between closely related analytes. It would be particularly desirable if such assays could distinguish between proteins, glycoproteins, carbohydrates, and other large biomolecules, which differ from each other in only very small ways, such as by a single amino acid deletion or substitution in a protein. Such methods and assays would be useful for distinguishing between the tissue and blood isoforms of CKMB in the diagnoses of acute myocardial infarction.
2. Description of the Background Art
U.S. Pat. Nos. 4,376,110 and 4,486,530, describe immunometric assays which employ a monoclonal capture antibody and a monoclonal detection antibody. Similar assays are performed using polyclonal antibodies as either or both of the capture and detection antibodies. Assays for diagnosing myocardial infarction based on the measurement of CKMB isoforms are described in Puleot et al. (1990) Circulation 82:759-764. Antibodies capable of binding to the M subunit and the B subunit of CKMB are described in U.S. Pat. No. 4,912,033.
The full disclosure of each of the references is incorporated herein by reference.